3. Cover beaker with watch glass and place in drying oven. Dry to 
constant weight at 95°C (about 48 hours). 
4. Cool and determine dry weight. 
5. Add concentrated nitric acid in sufficient quantity to cover 
organisms, adding acid in 20 ml increments. Cover beaker with 
watch glass. (Use same amount of acid for all samples within a 
group.) 
6. Let sample cold digest until tissue is well broken down; i.e., 4-24 
hours. 
7. Heat gently, to about 40°C, being careful to avoid frothing over. 
Continue until frothing stops. 
8. Heat to 85°C while covered, and bring sample to near dryness. (Be 
careful not to take to complete drymess at any time.) 
9. Remove from hot plate and add 20 ml of concentrated nitric acid. 
Repeat step 8. 
10. Repeat step 9 until digestion is complete, which is indicated by pale 
yellow color, clarification of the liquid, and no trace of lipids. 
(Treat all samples within a group in identical fashion.) 
11. Take to near dryness (about 5 ml remaining), cool, and add 20 ml 
of 5% nitric acid, getting all soluble residue into solution. 
12. Filter sample into 50 ml volumetric flask through Whatman #42 
Filter Paper which has been prerinsed with 5% nitric acid. 
13. Rinse beaker 2-3 times with 5% nitric acid and pour through filter. 
14. Rinse funnel down and bring up to volume with 5% nitric acid. 
15. Mix well, transfer to acid stripped 60 ml polyethylene bottle, and 
hold for A.A. analysis. 
NOTE: All glassware is detergent washed, soaked in 10% nitric acid, and 
copiously rinsed with deionized water. 
Each group of 1 5 samples is accompanied by a complete reagent methods 
blank. Analysis is performed on a Perkin-Elmer model 603 Atomic Absorption 
2 
